Polyazamacrocyclic compound, and a production method and a biomedical use therefor

ABSTRACT

According to the present invention, a novel polyazamacrocyclic compound which is used as a bifunctional chelating agent (BFC) can be synthesized selectively and in high yield. The novel polyazamacrocyclic compound synthesized by this method chelates with metals and thus can be conjugated with bioactive molecules such as peptides, and can be used in the diagnosis and treatment of medical conditions.

TECHNICAL FIELD

The present invention relates to novel polyazamacrocyclic compoundswhich are capable of chelating a bioactive molecule with metal ions as abifunctional chelator (BFC) and can be used for treatment and diagnosis,a method of preparation and biomedical use of the same.

BACKGROUND

Due to the ability of the macrocyclic molecules to coordinate withvarious metal cations, the discovery and synthesis oftetraazacycloalkane derivatives have attracted an increasing amount ofattention for the past few years. Among them, cyclen(1,4,7,10-tetraazacyclododecane) and cyclam(1,4,8,11-tetraazacyclotetradecane) have been the focus of research,where it has been found that their macrocyclic molecular structure isvery advantageous for forming metal complexes. Since such cyclicpolyamines exhibit strong affinity to certain metal ions where they arecapable of selectively binding with the metal ions, they can be used asmetal catalysts, reaction sites for methalloenzyme, cleavers forphosphoric esters such as DNA and RNA, radioactive diagnosis andtreatment, as well as MRI contrast agent, etc.

Among metal ions of high interest in the medical field, ions formingstable complexes with cyclen or cyclam derivatives include radioactiveisotopes which can be used in nuclear medicine, as well as Gd which canbe used as MRI contrast agent. ⁶⁴Cu, ¹¹¹In, ⁶⁷Ga, ⁸⁶Y, etc. areradioactive isotopes that can be used in diagnoses employing positronemission tomography (PET) or single photon emission computed tomography(SPECT), while ⁹⁰Y is a radioactive isotope that can be used for therapy[Anderson C J, Welch M J. Radiometal-Labeled Agents (Non-Technetium) forDiagnostic Imaging. Chem. Rev. 1999, 99, 2219-2234; Anderson C J, LewisJ S. Radiopharmaceuticals for targeted radiotherapy of cancer. ExpertOpinion on Therapeutic Patents 2000, 10, 1057-1069].

For instance, the use of radionuclides such as ⁶⁴Cu in nuclear medicineor preclinical applications has been on the rise, and BFC is used tosafely attach a radionuclide to a bioactive molecule, i.e. the targetmolecule. Thus, the development of BFC having excellent in vivostability is very critical in designing a system for delivering aradionuclide in vivo.

A great deal of effort has been made to develop a ligand which iscapable of chelating in a stable manner in vivo. The most common andgeneral BFCs that has been studied are DOTA(1,4,7,10-tetraazacyclododecan-1,4,7,10-tetracetic acid) and TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetracetic acid). However,recent studies show that such generally used BFCs are rather unstable invivo than the more recently developed BFCs such as cross-bridgedtetraamine ligands and sarcophagine ligands due to the increaseddissociation of metal.

Boswell et al. recently reported about cross-bridged cyclam derivativesfor peptide conjugation and ⁶⁴Cu radioactive labeling [C. AndrewBoswell. Celeste A. S. Regino, Kwamena E. Baidoo, Karen J. Wong, AmbikaBumb. Heng Xu, Diane E. Milenic, James A. Kelley. Christopher C. Lai.and Martin W. Brechbiel. Synthesis of a Cross-Bridged Cyclam Derivativefor Peptide Conjugation and ⁶⁴Cu Radiolabeling. Bioconjugate Chem. 2008,19, 1476-1484]. They synthesized a⁶⁴Cu-cross-bridged(CB)-TE2A(1,8-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane)-propeptidelinker, and conjugated c[RGDfK(s)]. Furthermore, the Archibald groupreported about NCSBz-CB-TE2A derivatives for bio-conjugation [ElizabethA. Lewis, Ross W. Boyle and Stephen J. Archibald, Ultrastable complexesfor in vivo use: a bifunctional chelator incorporating a cross-bridgedmacrocycle. Chem. Commun., 2004, 2212-2213]. However, the selectivefunctionalization of nitrogen in cyclic polyamines is not obvious, andis still a difficult task in the organic synthesis field. For example,the synthesis of NCSBz-CB-TE2A involves 13 steps including thepreparation of starting materials, and the overall yield of the finalproduct is only 8.7%. Therefore, there is a need to design novelpolyazamacrocyclic compounds that can be effectively used as BFCs, andto develop synthetic methods for preparing such compounds easily in ahigh yield.

DETAILED DESCRIPTION Technical Problem

The present invention is to overcome the above-mentioned problems ofconventional techniques. The object of the invention is to provide novelpolyazamacrocyclic compounds useful as BFC, to which varioussubstituents may be introduced and which can be easily synthesized inhigh yield, as well as a process for preparing the same.

Another object of the invention is to provide a method of using novelpolyazamacrocyclic compounds for biomedical use.

Technical Solution

In the present disclosure, a chelate means a compound in which amulti-dentate (at least bi-dentate) ligand is coordinated to a metalion. The ligand here is referred to as a chelator.

Further, a conjugate compound in the present disclosure indicates acompound where a chelator is bound to a protein, a peptide or anantibody via conjugation. A metal chelating conjugate compound means acompound where a chelate is bound to a protein, a peptide or an antibodyvia conjugation, or where a conjugate compound is bound to a metal ion(complex ion).

A pharmaceutical formulation for diagnosis or treatment according to thepresent invention is comprised by conjugation of a chelate of metalradionuclide to a target molecule such as a protein, a peptide, anantibody or an antibody fragment by means of a BFC. Thus, BFC contains areactive functional group such as an aromatic isothiocyanate group or anactivated ester, and reacts with a nucleophilic binding site such as—NH₂, —SH or —OH of the target molecule [Liu, S., and Edwards, D. S.Bifunctional chelators for therapeutic lanthanide radiopharmaceuticals.Bioconjugate Chem. 2001, 12, 7-34]. The activated ester may be activatedby those such as the functional groups shown below, but not limitedthereto.

In a pharmaceutical formulation for diagnosis or treatment according tothe present invention, a linker may be incorporated between the chelatorand the target molecule for the purpose of controlling thepharmacokinetic properties and distribution in vivo, if necessary[Parry, J. J., Kelly, T. S., Andrews, R., and Rogers, B. E. In vitro andin vivo evaluation of 64Cu-labeled DOTA-linkerbombesin (7-14) analoguescontaining different amino acid linker moieties. Bioconjugate Chem.2007, 18, 1110-1117; Dijkgraaf, I., Liu, S., Kruijtzer, J. A. W., Soede,A. C., Oyen, W. J. G., Liskamp, R. M. J., Corstens, F. H. M., andBoerman, O. C. Effects of linker variation on the in vitro and in vivocharacteristics of an 111In-labeled RGD peptide. Nucl. Med. Biol. 2007,34, 29-35; Li, L., Yazaki, P. J., Anderson, A.-L., Crow, D., Colcher,D., Wu, A. M., Williams, L. E., Wong, J. Y. C., Raubitschek, A., andShively, J. E. Improved biodistribution and radioimmunoimaging withpoly(ethylene glycol)-DOTA-conjugated anti-CEA diabody. BioconjugateChem. 2006, 17, 68-76]. Useful linkers include those represented by oneof the following chemical formulas, but not limited thereto.

A polyazamacrocyclic compound according to the present invention, or apharmaceutically acceptable salt thereof can be represented by ChemicalFormula 1:

-   -   wherein,    -   m, n, p and q are identical to or different from one another,        and individually represent an integer of 2 or 3,    -   r is an integer from 0 to 5,    -   t is an integer of 0 or 1,    -   r+t>0,    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are identical to or different        from one another, and individually represent H, C₁₋₅ alkyl or        C₃₋₆ cycloalkyl,    -   R¹⁰ represents H, C₁₋₅ alkyl, C₃₋₆ cycloalkyl, or C₇₋₁₄ aralkyl,    -   U and W are identical to or different from one another, and        individually represent H, C₁₋₅ alkyl or C₃₋₆ cycloalkyl,    -   Y and Z are identical to or different from one another, and        individually represent H, C₁₋₅ alkyl or C₃₋₆ cycloalkyl,    -   A represents C₆₋₁₀ aryl,    -   Q represents H, nitro, amino, isothiocyanato, maleimido, ester,        alkyne, aminoxy, thiol, azide or carboxylic acid.

According to the present invention, C₁₋₅ alkyl, C₃₋₆ cycloalkyl or C₇₋₁₄aralkyl of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁰, U, W, Y and Z may besubstituted with one or more substituent(s) selected from the groupconsisting of C₁₋₄ alkyl, halogen, hydroxyl, nitro, cyano, alkoxy,amino, ester and carboxylic group.

According to the present invention, C₆₋₁₀ aryl of A may be substitutedwith one or more substituent(s) selected from the group consisting ofC₁₋₄ alkyl, halogen, hydroxyl, alkoxy, ester and carboxylic group.

According to the present invention, the above pharmaceuticallyacceptable salt, when the compound represented by Chemical Formula 1contains a negatively charged component, comprises a cation or acationic group selected from the group consisting of potassium, sodium,lithium, ammonium, silver, calcium and magnesium, or when the compoundrepresented by Chemical Formula 1 contains a positively chargedcomponent, comprises an anion or an anionic group selected from thegroup consisting of F⁻, Cl⁻, Br⁻, I⁻, ClO₄ ⁻, BF₄ ⁻, HCO₃ ⁻, CH₃CO₂ ⁻,CH₃SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, CF₃SO₃ ⁻, H₂PO₄ ⁻ and B(C₆H₅)₄ ⁻, but are notlimited thereto.

Polyazamacrocyclic compounds or pharmaceutically acceptable saltsthereof according to the present invention serve as a BFC, and conjugateto a protein, a peptide, an antibody or an antibody fragment via anisothiocyanate group or an activated ester group.

Examples of polyazamacrocyclic compounds or pharmaceutically acceptablesalts thereof according to the present invention include1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecanerepresented by Chemical Formula 2,1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecanerepresented by Chemical Formula 3,1,7-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanerepresented by Chemical Formula 4,1,7-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,7,10-tetraazacyclododecanerepresented by Chemical Formula 5,1,8-bis-(carboxymethyl)-4-(4′-nitrophenethyl)-1,4,8,11-tetraazacyclotetradecanerepresented by Chemical Formula 6, and1,8-bis-(carboxymethyl)-4-(methyl)-1,4,8,11-tetraazacyclotetradecanerepresented by Chemical Formula 7.

Metals which chelate polyazamacrocyclic compounds or pharmaceuticallyacceptable salts according to the present invention may be radioactiveor non-radioactive, and selected from transition metals, lanthanideelements, actinide elements and metal main group elements. For example,chelating radioactive metals include ⁴³Sc, ⁴³V, ⁴⁴Sc, ⁴⁵Ti, ⁵¹Mn, ⁵¹Cr,⁵²Mn, ⁵²Fe, ⁵³Fe, ⁵⁵Co, ⁵⁶Co, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶²Zn,⁶³Zn, ⁶⁴Cu, ⁶⁵Zn, ⁶⁶Ga, ⁶⁶Ge, ⁶⁷Ge, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸CU, ⁶⁸Ga, ⁶⁹Ge, ⁶⁹As,⁷⁰As, ⁷⁰Se, ⁷¹Se, ⁷¹As, ⁷²As, ⁷³Se, ⁷⁴Kr, ⁷⁴Br, ⁷⁵Se, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,⁷⁷Kr, ⁷⁸Br, ⁷⁸Rb, ⁷⁹Rb, ⁷⁹Kr, ⁸¹Rb, ⁸²Rb, ⁸³Sr, ⁸⁴Rb, ⁸⁴Zr, ⁸⁵Y, ⁸⁶Y,⁸⁷Y, ⁸⁷Zr, ⁸⁸Y, ⁸⁹Zr, ⁹⁰Y, ⁸⁹Zr, ⁹²Tc, ⁹³Tc, ⁹⁴Tc, ⁹⁵Tc, ⁹⁵Ru, ⁹⁵Rh,⁹⁶Rh, ⁹⁷Rh, ⁹⁷Ru, ⁹⁸Rh, ⁹⁹Rh, ^(94m)Tc, ^(99m)Tc, ¹⁰⁰Rh, ¹⁰¹Ag, ¹⁰²Ag,¹⁰²Rh, ¹⁰³Ag, ¹⁰³Ru, ¹⁰⁴Ag, ¹⁰⁵Ag, ¹⁰⁵Ru, ¹⁰⁶Ag, ¹⁰⁸In, ¹⁰⁹In, ¹¹⁰In,¹¹¹In, ^(113m)In, ¹¹⁵Sb, ¹¹⁶Sb, ¹¹⁷Sb, ¹¹⁵Te, ¹¹⁶Te, ¹¹⁷Te, ¹¹⁷I, ¹¹⁸I,¹¹⁸Xe, ¹¹⁹Xe, ¹¹⁹I, ¹¹⁹Te, ¹²⁰I, ¹²⁰Xe, ¹²¹Xe, ¹²¹I, ¹²²I, ¹²³Xe, ¹²⁴I,¹²⁶I, ¹²⁸I, ¹²⁹La, ¹³⁰La, ¹³¹La, ¹³²La, ¹³³La, ¹³⁵La, ¹³⁶La, ¹⁴⁰Sm,¹⁴¹Sm, ¹⁴²Sm, ¹⁴⁴Gd, ¹⁴⁵Gd, ¹⁴⁵Eu, ¹⁴⁶Gd, ¹⁴⁶Eu, ¹⁴⁷Eu, ¹⁴⁷Gd, ¹⁴⁸Eu,¹⁴⁹Pr, ¹⁵⁰Eu, ¹⁵³Sm, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁰Au,¹⁹¹Au, ¹⁹²Au, ¹⁹³Au, ¹⁹³Tl, ¹⁹⁴Tl, ¹⁹⁴Au, ¹⁹⁵Tl, ¹⁹⁶Tl, ¹⁹⁷Tl, ¹⁹⁸Tl,²⁰⁰Tl, ²⁰⁰Bi, ²⁰¹Tl, ²⁰²Bi, ²⁰³Bi, ²⁰⁵Bi, ²⁰⁶Bi, ²¹¹As, ²¹²Bi or ²²⁵Ac,but are not limited thereto.

Suitable radioactive metals for SPECT include ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc and¹¹¹In; suitable radioactive metals for PET include ⁶⁰Cu, ⁶¹Cu, ⁶²Cu,⁶⁴Cu, ⁸⁶Y, ⁸⁹Zr and ^(94m)Tc; and suitable radioactive metals fortherapy include ⁶⁷Cu, ⁹⁰Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re [S. Liu,Bifunctional coupling agents for radiolabeling of biomolecules andtarget-specific delivery of metallic radionuclides, Advanced DrugDelivery Reviews 2008, 60, (2008) 1347-1370].

Among them, ⁶⁴Cu is a useful nuclide for PET imaging and targetedradioactive therapy due to its half-life (12.7 hours), decay property(β+ (19%), β− (39%)), and suitability in terms of productivity in alarge scale at high specificity by using a biomedical cyclotron.

The conjugate compounds according to the present invention includecompounds represented by Chemical Formula 1 or pharmaceuticallyacceptable salts thereof, which are conjugated with an amino acid, apeptide, a protein, a nucleoside, a nucleotide, an aptamer, a nucleicacid, an enzyme, a lipid, a nitrogen-containing vitamin, anitrogen-containing hormone, a medicine, a nanoparticle, an antibody oran antibody fragment.

The metal chelating conjugate compounds according to the presentinvention include compounds where the above chelate has been conjugatedwith an amino acid, a peptide, a protein, a nucleoside, a nucleotide, anaptamer, a nucleic acid, an enzyme, a lipid, a nitrogen-containingvitamin, a nitrogen-containing hormone, a medicine, a nanoparticle, anantibody or an antibody fragment; or a radioactive (in case of treatmentor diagnosis by nuclear medicine) or non-radioactive (in case ofcontrast media for MRI) metal ion, derived from, for example, transitionmetals, lanthanide elements, actinide elements or metal main groupelements has been bound to the above conjugate compound. The above metalchelating conjugate compounds are useful for treatment and diagnosis.

Contrast media according to the present invention include compoundsrepresented by Chemical Formula 1 or pharmaceutically acceptable saltsthereof. Specifically, using compounds represented by Chemical Formula 1or pharmaceutically acceptable salts thereof as a chelator, they can bechelated with a metal ion having paramagnetic property, such as Mn, Fe,and Gd, and conjugated with a pathognomonic bio-material, to be used as,for instance, a contrast media for sonogram, for computed tomography(CT), for magnetic resonance imaging (MRI), for treatment/diagnosis viaSPECT or PET.

Pharmaceutical formulations according to the present invention comprisethe above metal chelating conjugate compounds and pharmaceuticallyacceptable vehicles, and are used for the diagnosis and treatment oftumor, dementia or mycoplasma, pathogen surface antigens, toxins,enzymes, allergens, medicine, biologically active molecules, bacteria,fungi, viruses, parasites, diseases relating to the autoimmune, heart ornervous system. The pharmaceutical formulations according to the presentinvention are used for the diagnosis and treatment of tumors, inparticular.

Methods for diagnosing or treating a disease, a tumor for example, of amammal other than human involve administering an effective amount of theabove metal chelating conjugate compound to a mammal other than human.

Antibodies with which a chelator or a chelate according to the presentinvention would conjugate may be a monoclonal antibody or a polyclonalantibody, a chimeric antibody or a heteroantibody, or for example, anantibody containing a protein, which comprises a derivative of annexin,anti-CEA, tositumomab, trastuzumab, HUA33, epratuzumab, cG250,ibritumomab tiuxetan, or the like. Antibodies or antibody fragments thatcan be bound to the chelator or chelate according to the presentinvention may be prepared by techniques well known in the art.

Proteins with which a chelator or a chelate according to the presentinvention would conjugate may include for example albumin, TCII, HSA,annexin and Hb; peptides may include for example RGD-containing peptide,melanocyte-stimulating hormone (MSH) peptide, neurotensin, calcitonin,threotide, bombesin, neurotensin, urotensin II and angiotensin;nitrogen-containing vitamins may include for example vitamin A, B1, B2,B12, C, D2, D3, E, H and K; and nitrogen-containing hormones may includefor example estradiol, progesterone and testosteron; but are not limitedthereto.

The method of preparing the polyazamacrocyclic compound represented byChemical Formula 1 or Chemical Formula 8 according to the presentinvention is described as follows:

wherein, m, n, p, q, r, t, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁰, U, W, Y,Z, A and Q are defined as in Chemical Formula 1.

The method of preparing a polyazamacrocyclic compound represented byChemical Formula 1 or 8 according to the present invention involves thesteps of (i) reacting a compound represented by Chemical Formula 9 withα-halocarboxylic ester (X—CUW—CO₂R⁹) to obtain atrans-N,N′-disubstituted compound represented by Chemical Formula 10,(ii) reacting the compound represented by Chemical Formula 10 with abase to obtain a compound represented by Chemical Formula 11, and (iii)incorporating a functional group —(CYZ)_(r)-A_(t)-Q [wherein, r, t, Y,Z, A and Q are defined as in Chemical Formula 1] to a secondary aminegroup in the cycle of compound represented by Chemical Formula 11 toform a compound represented by Chemical Formula 1 or 8:

wherein

m, n, p, q, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U and W are defined as inChemical Formula 1,

R⁹ represents C₁₋₅ alkyl, C₃₋₆ cycloalkyl, or C₇₋₁₄ aralkyl, and

X represents F, Cl, Br or I.

In the compounds according to the present invention, C₁₋₅ alkyl, C₃₋₆cycloalkyl or C₇₋₁₄ aralkyl of R⁹ may be substituted with one or moresubstituent(s) selected from the group consisting of C₁₋₄ alkyl,halogen, hydroxyl, nitro, cyano, alkoxy, amino, ester and carboxylicgroup.

In the method of preparing a polyazamacrocyclic compound represented byChemical Formula 1 or 8 according to the present invention, the reactionfor introducing Q can be carried out according to a method well known inthe art. For example, if Q of said Chemical Formula is H, step (iii)involves reacting the compound of Chemical Formula 11 withX—(CYZ)_(r)-A_(t)-H (X is defined as in Chemical Formula 10) to providethe compound of Chemical Formula 12 or 13. If Q of said Chemical Formulais nitro, amino, isothiocyanato or maleimido, step (iii) involvesreacting the compound of Chemical Formula 11 with X—(CYZ)_(r)-A_(t)-NO₂(X is defined as in Chemical Formula 10) to provide the compound ofChemical Formula 14 or 15.

wherein

m, n, p, q, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, W, Y, Z and A are definedas in Chemical Formula 1, and R⁹ and X are defined as in ChemicalFormula 10.

In the method of preparing a polyazamacrocyclic compound represented byChemical Formula 1 or 8 according to the present invention, theadditional reaction to introduce Q into the compound of Chemical Formula14 or 15 can be carried out according to a method well known in the art.For example, if Q of said Chemical Formula is amino, step (iii) furtherinvolves the step of reducing the nitro group of the compound ofChemical Formula 14 or 15 to an amine group. If Q of said ChemicalFormula is isothiocyanato, step (iii) further involves the step ofreducing the nitro group of the compound of Chemical Formula 14 or 15 toan amine group, which is then reacted with thiophosgen. If Q ismaleimido, step (iii) further involves the step of reducing the nitrogroup of the compound of Chemical Formula 14 or 15 to an amine group,which is then reacted with maleic anhydride.

Reaction of conventional cyclens or cyclams with 2 equivalents of alkylor aryl halide form mixtures of monosubstituted-, disubstituted-, andeven trisubstituted-macrocyclic molecules. Furthermore, depending on therelative position of the pendent arms, there would be three types ofN,N′-disubstituted cyclic polyamines, i.e. two types ofcis-disubstituted derivatives and one type of trans-disubstitutedderivative. Among them, trans-N,N′-disubstituted cyclen and cyclam areparticularly remarkable, because they can derive a stable 6-coordinatedcompound during chelate formation. Besides, trans-N,N′-double protectedcyclen and cyclam are convenient precursors for synthesizingthree-dimensional systems such as cryptands (cyclic polyether polyamine)based on cyclens or cyclams.

According to the present invention, a bisaminal compound (compound 9) isprepared as the starting material, which is then reacted withα-halocarboxylic ester (X—CUW—CO₂R⁹) to obtain atrans-N,N′-disubstituted polyazamacrocyclic compound or derivativesthereof (compound 1, 8, 10 to 15) easily and with high yield.

In the present invention, R⁹ as a protective group for carboxylic acidmay be C₁₋₅ alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyland tert-butyl, C₃₋₆ cycloalkyl such as cyclopentyl and cyclohexyl,C₆₋₁₂ aryl such as phenyl and α-naphthyl, phenyl-C₁₋₂ alkyl such asbenzyl and phenethyl, C₇₋₁₄ aralkyl such as α-naphthyl-C₁₋₂ alkyl (e.g.α-naphthylmethyl), or silyl. The protective group may be substitutedwith one or more substituent(s) from C₁₋₄ alkyl, halogen, hydroxyl,nitro, cyano, alkoxy, amino, ester and carboxyl group, but are notlimited thereto.

Among them, benzyl and tert-butyl as protective groups for thecarboxylic group are desirable since they are stable in a basicenvironment but are easily removable under acidic conditions. In thisregard, tert-butylbromoacetate or benzyl bromoacetate, for example, isused as α-halocarboxylic ester.

In order to remove the protective group for carboxylic groups, anyconventional method can be used including, for example, reduction oracidolysis processes.

The above reduction process may include contact reduction usingcatalysts such as Pd/C, palladium black and platinum oxide, reduction bysodium in liquid ammonium, or reduction by means of dithiothreitol. Theabove acidolysis process may include acidolysis by means of an inorganicacid such as hydrogen fluoride, hydrogen bromide and hydrogen chloride,or an organic acid such as trifluoroacetic acid, methanesulfonic acidand trifluoromethanesulfonic acid, or a mixture thereof.

Inert solvents used for the method of preparation according to thepresent invention may include water, methanol, ethanol, isopropanol,isobutyl alcohol, tert-butyl alcohol, acetonitrile (MeCN),tetrahydrofuran (THF), chloroform (CHCl₃), dimethylformamide (DMF),dimethylsulfoxide (DMSO), benzene, toluene, xylene, dichloromethane,ethylene glycol, acetone, n-propyl ketone, trichloroethylene, ether,cyclohexanone, butyrolactone or a mixture thereof, but are not limitedthereto.

In the method of preparation according to the present invention, thebase used for the basic hydrolysis of compound represented by ChemicalFormula 10 may include KOH, NaOH, Ca(OH)₂, Li[NTf₂], KF/Al₂O₃ and thelike, but are not limited thereto.

The trans-N,N′-disubstitution according to the present invention iscarried out at an ambient temperature, but may be carried out attemperatures higher or lower than that, if necessary.

The reaction times for each step in the method of preparation accordingto the present invention are generally from 1 hour to 5 days,specifically from 3 hours to 2 days, but may be longer or shorter thanthat, if necessary.

Advantageous Effects

The polyazamacrocyclic compounds according to the present invention canbe easily synthesized and conveniently purified in high yield whileminimizing separation by chromatography which requires intensive timeand labor.

According to the present invention, trans-N,N′-disubstituted cyclicpolyamine can be selectively synthesized by reacting bisaminal andα-halocarboxylic ester.

The polyazamacrocyclic compounds according to the present invention actas a useful BFC, and can be applied in the biomedical field, for examplefor radioactive labeling of target molecules such as peptides, bychelating with a radionuclide such as ⁶⁴Cu.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mass spectrum of TE2A-NO₂ according to an Example.

FIG. 2 shows a mass spectrum of Cu-TE2A-NO₂ according to an Example.

FIGS. 3A and 3B show natural log of absorbance vs. time plots of Cu-TE2A(Comparative Example) and Cu-TE2A-NO₂ (Example), respectively from anexperiment of acid decomplexation monitored by a UV-VISspectrophotometer.

FIGS. 4A and 4B show the results of measured cyclic voltammograms ofCu-TE2A (Comparative Example) and Cu-TE2A-NO₂ (Example), respectively.

FIG. 5 shows a mass spectrum of TE2A-mono-methyl to which tert-butyl hasbeen introduced as a protective group for carboxylic acid, according toan Example.

FIG. 6 shows a mass spectrum of TE2A-mono-methyl from which theprotective group for carboxylic acid has been removed, according to anExample.

FIG. 7 shows a mass spectrum of TE2A-di-methyl to which tert-butyl hasbeen introduced as a protective group for carboxylic acid, according toan Example.

FIG. 8 shows a mass spectrum of TE2A-di-methyl from which the protectivegroup for carboxylic acid has been removed, according to an Example.

FIG. 9 shows a mass spectrum of Cu-TE2A-mono-methyl according to anExample.

FIG. 10 shows a mass spectrum of Cu-TE2A-di-methyl according to anExample.

FIG. 11 shows a semi-preparative HPLC chromatogram of TE2A-c(RGDyK)according to an Example.

FIG. 12 shows a HPLC chromatogram for analysis of TE2A-c(RGDyK)according to an Example.

FIG. 13 shows a mass spectrum of TE2A-c(RGDyK) according to an Example.

FIG. 14 shows a radio-TLC chromatogram of ⁶⁴Cu-TE2A-c(RGDyK) metalchelating conjugate compound in which TE2A-c(RGDyK) has been labeledwith ⁶⁴Cu, according to an Example.

FIG. 15 shows a HPLC radio chromatogram for analysis of⁶⁴Cu-TE2A-c(RGDyK) metal chelating conjugate compound in whichTE2A-c(RGDyK) has been labeled with ⁶⁴Cu, according to an Example.

FIG. 16 shows HPLC chromatograms for analysis of both TE2A-c(RGDyK)conjugate compound according to an Example and of ⁶⁴Cu-TE2A-c(RGDyK)metal chelating conjugate compound (in which TE2A-c(RGDyK) has beenlabeled with ⁶⁴Cu), respectively, in order to confirm the preparation ofthe metal chelating conjugate compound.

FIG. 17 shows the result of the in vivo distribution test of⁶⁴Cu-TE2A-c(RGDyK) metal chelating conjugate compound in whichTE2A-c(RGDyK) has been labeled with ⁶⁴Cu, according to an Example.

FIG. 18 illustrates administering ⁶⁴Cu-TE2A-c(RGDyK) metal chelatingconjugate compound in which TE2A-c(RGDyK) has been labeled with ⁶⁴Cu,according to an Example, to a female nude mouse having U87MG tumorcells.

FIG. 19 shows a micro-PET image with the lapse of time afteradministering ⁶⁴Cu-TE2A-c(RGDyK) metal chelating conjugate compound inwhich TE2A-c(RGDyK) has been labeled with ⁶⁴Cu, according to an Example,to a female nude mouse having U87MG tumor cells.

FIG. 20 shows a radio-ITLC chromatogram of ⁶⁴Cu-TE2A-trastuzumab metalchelating conjugate compound in which TE2A-trastuzumab has been labeledwith ⁶⁴Cu, according to an Example.

FIG. 21 shows SEC HPLC chromatograms of both TE2A-trastuzumab conjugatecompound according to an Example and of ⁶⁴Cu-TE2A-trastuzumab metalchelating conjugate compound (in which TE2A-trastuzumab has been labeledwith ⁶⁴Cu), respectively, in order to confirm the preparation of themetal chelating conjugate compound.

FIG. 22 shows the result of the in vivo distribution test of⁶⁴Cu-TE2A-trastzumab metal chelating conjugate compound in whichTE2A-trastuzumab has been labeled with ⁶⁴Cu, according to an Example.

FIG. 23 illustrates administering ⁶⁴Cu-TE2A-trastuzumab metal chelatingconjugate compound in which TE2A-trastuzumab has been labeled with ⁶⁴Cu,according to an Example, to a female nude mouse having NIH3T6.7 tumorcells.

FIG. 24 shows a micro-PET image with the lapse of time afteradministering ⁶⁴Cu-TE2A-trastuzumab metal chelating conjugate compoundin which TE2A-trastuzumab has been labeled with ⁶⁴Cu, according to anExample, to a female nude mouse having NIH3T6.7 tumor cells.

DETAILED DESCRIPTION

The present invention is described in further detail by ReferenceExamples and Examples provided below. However, those Reference Examplesand Examples are merely for illustration to help understand the presentinvention, of which the scope is not limited thereto.

Reference Examples 1˜8

By employing tert-butylbromoacetate and benzyl bromoacetate as anα-halocarboxylic ester (X—CUW—CO₂R⁹), the conditions for reaction withcompound (1) of Reaction Scheme (1) were examined.

Specifically, as shown in Table 1 below, trans-N,N′-disubstituted cyclamwas synthesized by varying the types of solvent and equivalent amountsof α-halocarboxylic ester.

TABLE 1 Ref. Ex. Reactants Equivalent Solvent Yield (%) 1tert-Butylbromoacetate 4 CH₃CN 95 2 tert-Butylbromoacetate 2 CH₃CN 62 3tert-Butylbromoacetate 4 THF 40 4 tert-Butylbromoacetate 4 CHCl₃ 45 5Benzyl bromoacetate 4 CH₃CN 88 6 Benzyl bromoacetate 2 CH₃CN 52 7 Benzylbromoacetate 4 THF 42 8 Benzyl bromoacetate 4 CHCl₃ 46 Reactionconditions: compound (1) = 0.325 g (1.44 mmol), solvent = 20 ml, ambienttemperature, 24 hours

As shown in Table 1 above, the most effective result of 95% yield wasobtained when tert-butylbromoacetate was used as an alkylating agent,and CH₃CN (MeCN) as a solvent (Reference Example 1 of Table 1). Whenusing THF and CHCl₃, moderate yields (40%, 45%) were obtained,respectively (Reference Examples 3 and 4 of Table 1). The amount oftert-butylbromoacetate used for the reaction was 2 equivalents or 4equivalents, and better selectivity and yield were obtained when using 4equivalents of tert-butylbromoacetate (Reference Examples 1 and 2 ofTable 1).

Examples 1 and 2

By using tert-butylbromoacetate and benzyl bromoacetate asα-halocarboxylic ester (X—CUW—CO₂R⁹), TE2A(1,8-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane (compound(6)) was synthesized according to the route illustrated by the followingReaction Scheme:

Example 1 Preparation of TE2A (6) using benzyl 2-bromoacetatePreparation of 1,4,8,11-tetraazacyclo[9.3.1.1]-hexadecane (1)

Compound (1) was prepared according to a modified procedure which waspreviously reported by R. Guilard, C. Lecomte et al. in a amplifiedscale. In short, 2 equivalents of formaldehyde (15.1 ml, 37% in water)were rapidly added to an aqueous solution of cyclam (20.3 g, 0.10 M in200 ml of distilled water) at a temperature of 0˜5° C. After warming thereaction mixture to an ambient temperature, it was stirred for 2 hours.Then the reaction mixture was cooled to a temperature of 0 to 5° C., anda white precipitate thus generated was filtered and washed with coldwater (2×10 ml). The obtained white solid was dissolved in CHCl₃ (200ml) and dried over MgSO₄. Chloroform was evaporated under reducedpressure to obtain compound (1) (20.95 g, 92% yield). The spectrometricdata of compound (1) exactly matched those reported previously. ¹H NMR(400 MHz, CDCl₃): δ 5.63-5.60 (dt, 2H, J=10.8 Hz), 3.14-3.12 (d, 4H,J=9.8 Hz), 2.90-2.87 (d, 2H, J=10.8 Hz), 2.84-8.80 (m, 4H), 2.65-2.58(m, 4H), 2.38-2.35 (d, 4H, J=9.9 Hz), 2.3-2.1 (m, 2H), 1.17-1.14 (m,2H); ¹³C NMR (100.6 MHz, CDCl₃): δ 69.3, 54.1, 49.8, 20.6.

Preparation of1,8-bis-(benzyloxycarbonylmethyl)-4,11-diazoniatricyclo[9.3.1.1]hexadecanedibromide (2)

Four equivalents of benzyl 2-bromoacetate (10.29 ml, 15.03 g, 65.6 mmol)were added to a portion of a stirred solution of compound (1) (3.68 g,16.40 mmol) in MeCN (100 ml). The reaction mixture was stirred at anambient temperature for 24 hours. A yellowish white precipitate thusgenerated was filtered and washed with MeCN (2×20 ml), and dried invacuo. The crude product was recrystallized from ethanol to obtaincompound (2) as a white solid (10.3 g, 92% yield). ¹H NMR (500 MHz,DMSO-d₆): δ 7.32-7.41 (m, 10H, ArH), 5.16 (s, 4H), 3.52 (s, 4H), 3.33(s, 4H), 3.09 (brs, 8H), 2.85 (brs, 4H), 2.76-2.74 (t, 4H, J=5 Hz), 1.86(brs, 4H); ¹³C NMR (125 MHz, DMSO-d₆): δ 172.2, 135.5, 128.4, 128.2,128.0, 66.4, 55.9, 54.0, 52.8, 51.2, 47.3, 44.1, 22.1, 18.5; HRMS (ESI)calculated for C₃₀H₄₂N₄O₄: 523.3284 [(M+H)⁺], measured value: 523.3281[(M+H)⁺].

Preparation of1,8-bis-(benzyloxycarbonylmethyl)-1,4,8,11-tetraazacyclotetradecane (4)

To compound (2) (9.23 g, 13.52 mmol) was added a 3 M NaOH solution (200ml). After stirring for 3 hours, the resultant solution was extractedwith CHCl₃ (3×100 ml), and the combined organic layer was washed withbrine and dried over MgSO₄. Evaporation of the solvent under reducedpressure gave oil, which was then solidified to obtain compound (4)(6.58 g, 98% yield). ¹H NMR (500 MHz, CDCl₃): δ 7.20-7.14 (m, 10H, ArH),4.92 (s, 4H), 3.25 (s, 4H), 2.71-2.66 (m, 12H), 2.49-2.47 (t, 4H, J=4.2Hz), 1.70 (brs, 4H); ¹³C NMR (125 MHz, CDCl₃): δ 171.3, 135.0, 128.3,128.1, 127.8, 66.2, 54.7, 54.0, 51.9, 49.1, 46.3, 24.3; HRMS (FAB)calculated for C₂₈H₄₁N₄O₄: 497.3128 [(M+H)⁺], measured value: 497.3129[(M+H)⁺].

Preparation of 1,8-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane(TE2A) (6)

To a solution of compound (4) (0.48 g, 0.96 mmol) in absolute ethanol(40 ml) was added 10% Pd/C (0.12 g). The resultant mixture was stirredat an ambient temperature under H₂ atmosphere for 10 hours. The reactionmixture was filtered through a celite pad, and washed with ethanol (2×10ml). The combined filtrate was evaporated in vacuo to give an oilyresidue, which was then treated with diethyl ether (Et₂O) to obtain anoff-white solid (0.29 g, 98% yield). ¹H NMR (500 MHz, D₂O): δ 3.48 (brs,2H), 3.01-3.19 (m, 10H), 2.80 (brs, 6H), 2.67 (brs, 2H), 1.84 (brs, 4H);¹³C NMR (125 MHz, D₂O): δ 179.0, 56.3, 55.7, 48.9, 45.4, 22.8; HRMS(FAB) calculated for C₁₄H₂₈N₄O₄: 317.2189 [(M+H)⁺], measured value:317.2185 [(M+H)⁺].

Example 2 Preparation of TE2A (6) using tert-butylbromoacetatePreparation of1,8-bis-(carbo-tert-butoxymethyl)-4,11-diazoniatricyclo[9.3.1.1]hexadecanedibromide (3)

Four equivalents of tert-butylbromoacetate (9.38 ml, 12.38 g, 63.48mmol) were added to a portion of a stirred solution of compound (1)(3.56 g, 15.87 mmol) in MeCN (100 ml). The reaction mixture was stirredat an ambient temperature for 24 hours. A yellowish white precipitatethus generated was filtered and washed with MeCN (2×20 ml), and dried invacuo. The crude product was recrystallized from ethanol to obtaincompound (3) as white solid (9.26 g, 95% yield). ¹H NMR (500 MHz,DMSO-d₆): δ 1.48 (s, 18H), 1.76-1.78 (d, 2H, J=8.5 Hz), 2.35-2.45 (m,4H), 2.70-2.73 (d, 2H, J=15 Hz), 3.08-3.09 (d, 2H, J=5 Hz), 3.24-3.38(m, 4H), 3.53-3.58 (m, 2H), 3.64-3.66 (d, 2H, J=10 Hz), 3.79-3.81 (d,2H, J=11.5 Hz), 4.33-4.38 (t, 2H, J=14 Hz), 4.43-4.46 (d, 2H, J=16.5Hz), 4.59-4.62 (d, 2H, J=16.5 Hz), 5.23-5.25 (d, 2H, J=9.5 Hz); ¹³C NMR(125 MHz, DMSO-d₆): δ 163.5, 84.2, 76.5, 59.8, 57.2, 50.6, 47.7, 46.3,27.5, 19.2; HRMS (ESI) calculated for C₂₄H₄₇N₄O₄: 455.3591 [(M+H)⁺],measured value: 455.3594 [(M+H)⁺].

Preparation of1,8-bis-(carbo-tert-butoxymethyl)-1,4,8,11-tetraazacyclotetradecane (5)

To compound (3) (9.15 g, 14.89 mmol) was added a 3 M NaOH solution (200ml). After stirring for 3 hours, the resultant solution was extractedwith CHCl₃ (3×100 ml), and the combined organic layer was washed withbrine and dried over MgSO₄. Evaporation of the solvent under reducedpressure gave oil, which was then solidified to obtain compound (5)(6.25 g, 98% yield). ¹H NMR (500 MHz, CDCl₃): δ 3.25 (s, 4H), 2.72-2.59(m, 16H), 1.71-1.69 (m, 4H), 1.37 (s, 18H); ¹³C NMR (125 MHz, CDCl₃): δ170.43, 80.57, 54.74, 54.13, 52.47, 50.02, 47.59, 28.09, 25.78; HRMS(FAB) calculated for C₂₂H₄₅N₄O₄: 429.3441 [(M+H)⁺], measured value:429.3439 [(M+H)⁺].

Preparation of 1,8-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane(TE2A.2TFA) (6.2TFA)

Compound (5) (1.12 g, 2.61 mmol) was dissolved in a mixture of CF₃CO₂H(TFA) and CH₂Cl₂ (1:1 (v/v), 40 ml). The resultant mixture was stirredat an ambient temperature for 24 hours. The solvent was evaporated underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain an off-white solid of compound (6) (1.39 g, 98% yield,calculated as 2 equivalents of TFA for the basic weight). ¹H NMR (500MHz, D₂O): δ 3.31 (brs, 2H), 3.22-2.95 (m, 8H), 2.94-2.72 (m, 8H), 2.64(brs, 2H), 1.83 (brs, 4H); ¹³C NMR (125 MHz, D₂O): δ 180.9, 57.5, 56.9,55.4, 49.2, 46.2, 23.7; HRMS (FAB) calculated for C₁₄H₂₈N₄O₄: 317.2189[(M+H)⁺], measured value: 317.2185 [(M+H)⁺].

Examples 3 and 4

TE2A-NCS, to which different isothiocyanates have been introduced andfunctionalized, was prepared from compound (5) obtained from Example 2,via either one of the two routes shown in Reaction Scheme 3 below.

Example 3 Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecane(13) using tert-butylbromoacetate Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4-(4′-nitrobenzyl)-1,4,8,11-tetraazacyclotetradecane(7)

To a solution of compound (5) (1.27 g, 2.96 mmol) in dry CHCl₃ (50 ml)were added triethylamine (1.21 ml, 0.90 g, 8.88 mmol) and 4-nitrobenzylbromide (0.64 g, 2.96 mmol). After stirring at an ambient temperaturefor 10 hours, the solvent was removed under reduced pressure, and theresidue was purified through column chromatography on alumina (basic).Extraction with a mixture of ethyl acetate and methanol (10:2) gave apurified clear oil, which was then solidified to obtain compound (7)(1.30 g, 78% yield). ¹H NMR (500 MHz, CDCl₃): δ 8.11-8.10 (dd, 2H),7.55-7.53 (dd, 2H), 3.56 (s, 2H), 3.24 (s, 2H), 3.20-3.06 (m, 6H), 3.01(brs, 2H), 2.70-2.58 (m, 4H), 2.54-2.42 (m, 4H), 2.39 (brs, 2H), 1.93(brs, 2H), 1.75 (brs, 2H), 1.39-1.36 (dd, 18H); ¹³C NMR (125 MHz,CDCl₃): δ 171.3, 170.5, 147.0, 146.6, 130.1, 123.3, 81.9, 81.2, 58.1,56.4, 55.8, 54.9, 53.1, 51.6, 51.5, 51.2, 49.6, 48.5, 46.1, 28.0, 25.1,22.7; HRMS (FAB) calculated for C₂₉H₅₀N₅O₆: 564.3761 [(M+H)⁺], measuredvalue: 564.3757 [(M+H)⁺].

Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4-(4′-aminobenzyl)-1,4,8,11-tetraazacyclotetradecane(9)

To a solution of compound (7) (1.22 g, 2.16 mmol) in absolute ethanol(100 ml) was added 5% Pd/CaCO₃ (0.31 g) to which lead (Pb) had beenadded as an inhibitor. The resultant mixture was stirred at an ambienttemperature under H₂ atmosphere for 12 hours. The reaction mixture wasfiltered through a celite pad and washed with ethanol (2×20 ml). Thecombined filtrate was evaporated in vacuo to give an oily residue, whichwas then treated with Et₂O to obtain an off-white solid of compound (9)(1.13 g, 98% yield). ¹H NMR (500 MHz, CDCl₃): δ 7.07-7.06 (dd, 2H),6.67-6.65 (dd, 2H), 3.39 (s, 4H), 3.30-3.14 (m, 4H), 3.12-3.01 (m, 4H),2.77-2.75 (m, 2H), 2.70-2.42 (m, 8H), 1.96 (brs, 2H), 1.80 (brs, 2H),1.46-1.45 (dd, 18H); ¹³C NMR (125 MHz, CDCl₃): δ 171.1, 170.7, 145.6,130.7, 127.6, 115.0, 81.6, 81.2, 57.9, 55.4, 54.7, 54.1, 51.6, 51.5,49.8, 49.7, 49.4, 46.0, 28.2, 24.3, 24.1, 22.5; HRMS (FAB) calculatedfor C₂₉H₅₂N₅O₄: 534.4019 [(M+H)⁺], measured value: 534.4024 [(M+H)⁺].

Preparation of1,8-bis-(carboxymethyl)-4-(4′-aminobenzyl)-1,4,8,11-tetraazacyclotetradecane.2TFA(11.2TFA)

Compound (9) (0.92 g, 1.72 mmol) was dissolved in a mixture of TFA andCH₂Cl₂ (1:1 (v/v), 28 ml). The resultant mixture was stirred at anambient temperature for 24 hours. The solvent was evaporated underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain an off-white solid of compound (11) (1.11 g, 99% yield,calculated as 2 equivalents of TFA for the basic weight). ¹H NMR (500MHz, D₂O): δ 7.20-7.10 (dd, 2H), 6.83-6.71 (dd, 2H), 4.1 (s, 2H),3.52-2.42 (m, 20H), 2.1-1.62 (m, 4H); ¹³C NMR (125 MHz, D₂O): δ 192.7,180.5, 180.4, 149.5, 148.2, 145.3, 133.1, 129.8, 128.8, 117.76, 116.4,116.2, 114.0, 58.3, 57.1, 56.5, 55.0, 51.1, 48.8, 46.5, 45.8, 45.4,23.7, 23.3, 22.7; HRMS (FAB) calculated for C₂₁H₃₆N₅O₄: 422.2767[(M+H)⁺], measured value: 422.2768 [(M+H)⁺].

Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecane-2TFA(13.2TFA)

To a solution of compound (11) (0.98 g, 1.51 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (3.47 ml, 5.21 g, 45.30 mmol)in CHCl₃ (10 ml). The reaction mixture was stirred at an ambienttemperature for five hours to separate the layers. After removing theaqueous layer, the organic CHCl₃ layer was washed with water (2×50 ml).The combined aqueous layer was washed with CHCl₃ (3×50 ml) to remove theunreacted thiophosgene. Finally, the aqueous layer was lyophilized toobtain a white solid of compound (13) (1.02 g, 98% yield). ¹H NMR (500MHz, D₂O): δ 7.66-7.64 (dd, 2H), 7.49-7.47 (dd, 2H), 4.03 (s, 2H),3.50-2.51 (m, 20H), 2.09 (brs, 2H), 1.87 (brs, 2H); ¹³C NMR (125 MHz,D₂O): δ 192.6, 176.2, 175.7, 133.7, 132.8, 132.0, 123.9, 56.4, 56.2,54.5, 54.0, 51.4, 50.6, 50.2, 49.9, 49.28, 47.7, 45.0, 22.6, 21.7, 13.4;HRMS (FAB) calculated for C₂₂H₃₄N₅O₄S: 464.2332 [(M+H)⁺], measuredvalue: 464.2329 [(M+H)⁺].

Example 4 Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecane(14) using tert-butylbromoacetate Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4-(4′-nitrophenethyl)-1,4,8,11-tetraazacyclotetradecane(8)

A solution of compound (5) (1.37 g, 3.19 mmol), 4-nitrophenethyl bromide(1.47 g, 6.38 mmol), anhydrous K₂CO₃ (1.32 g, 9.57 mmol) and KI (1.59 g,9.57 mmol) dissolved in dry toluene (150 ml) was stirred under refluxfor 24 hours. The solvent was evaporated from the reaction mixture underreduced pressure, and CH₂Cl₂ (250 ml) was added thereto. The resultantbrown slurry was filtered through a celite pad, and washed with CH₂Cl₂(2×30 ml). The solvent was evaporated from the combined filtrate underreduced pressure. The residue thus obtained was purified via alumina(basic) column chromatography using EtOAc/methanol (10:2) as an eluentto provide compound (8) as a yellow oil (1.26 g, 68% yield). ¹H NMR (500MHz, CDCl₃): δ 8.07-8.06 (dd, 2H), 7.35-7.33 (dd, 2H), 3.23-3.20 (dd,4H), 2.97 (brs, 4H), 2.87-2.82 (m, 4H), 2.71-2.51 (m, 12H), 1.88 (brs,2H), 1.61 (brs, 2H), 1.39-1.37 (dd, 18H); ¹³C NMR (125 MHz, CDCl₃): δ170.7, 170.5, 148.6, 146.3, 129.5, 123.5, 81.3, 81.1, 55.7, 55.3, 55.0,52.5, 52.0, 50.2, 49.5, 48.4, 46.1, 32.0, 28.1, 24.4, 24.3, 23.2; HRMS(FAB) calculated for C₃₀H₅₂N₅O₆: 578.3918 [(M+H)⁺], measured value:578.3915 [(M+H)⁺].

Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4-(4′-aminophenethyl)-1,4,8,11-tetraazacyclotetradecane(10)

To a solution of compound (8) (1.15 g, 1.99 mmol) in absolute ethanol(100 ml) was added 5% Pd/CaCO₃ (0.31 g) to which lead (Pb) had beenadded as an inhibitor. The resultant mixture was stirred at an ambienttemperature under H₂ atmosphere for 12 hours. The reaction mixture wasfiltered through a celite pad and washed with ethanol (2×20 ml). Thesolvent was evaporated from the combined filtrate in vacuo to give anoily residue, which was then treated with Et₂O to obtain a white solidof compound (10) (1.09 g, 98% yield). ¹H NMR (500 MHz, CDCl₃): δ6.90-6.88 (dd, 2H), 6.55-6.54 (dd, 2H), 3.28-3.25 (dd, 4H), 2.96-2.94(m, 2H), 2.88-2.86 (m, 2H), 2.81-2.79 (m, 2H), 2.70-2.68 (m, 2H),2.64-2.62 (m, 2H), 2.56 (brs, 8H), 2.47 (brs, 2H), 1.86 (brs, 2H), 1.60(brs, 2H), 1.39-1.37 (dd, 18H); ¹³C NMR (125 MHz, CDCl₃): δ 178.1,170.5, 170.4, 144.5, 129.9, 129.2, 115.1, 81.1, 80.9, 55.3, 55.0, 54.8,52.0, 51.7, 51.3, 50.4, 48.6, 48.3, 48.22, 45.8, 30.9, 28.1, 24.4, 23.8,22.8; HRMS (FAB) calculated for C₃₀H₅₄N₅O₄: 548.4176 [(M+H)⁺], measuredvalue: 548.4172 [(M+H)⁺].

Preparation of1,8-bis-(carboxymethyl)-4-(4′-aminophenethyl)-1,4,8,11-tetraazacyclotetradecane.2TFA(12.2TFA)

Compound (10) (0.95 g, 1.73 mmol) was dissolved in a mixture of TFA andCH₂Cl₂ (1:1 (v/v), 28 ml). The resultant mixture was stirred at anambient temperature for 24 hours. The solvent was evaporated underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain a white solid of compound (12) (1.14 g, 99% yield,calculated as 2 equivalents of TFA for the basic weight). ¹H NMR (500MHz, D₂O): δ 7.45-7.43 (dd, 2H), 7.38-7.36 (dd, 2H), 3.49-3.12 (m, 18H),2.82-2.62 (m, 6H), 1.91 (brs, 4H); ¹³C NMR (125 MHz, D₂O): δ 177.0,176.6, 163.4, 163.1, 162.8, 162.5, 137.4, 130.4, 128.9, 123.4, 119.9,117.5, 115.2, 112.9, 56.0, 54.9, 54.4, 53.3, 51.6, 50.5, 48.8, 47.3,45.0, 31.5, 27.6, 22.8, 21.3; HRMS (FAB) calculated for C₂₂H₃₈N₅O₄:436.2924 [(M+H)⁺], measured value: 436.2925 [(M+H)⁺].

Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecane-2TFA(14.2TFA)

To a solution of compound (12) (1.05 g, 1.58 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (3.63 ml, 5.45 g, 47.40 mmol)in CHCl₃ (10 ml). The reaction mixture was stirred at an ambienttemperature for 5 hours to separate the layers. After removing theaqueous layer, the organic CHCl₃ layer was washed with water (2×50 ml).The combined aqueous layer was washed with CHCl₃ (3×50 ml) to remove theunreacted thiophosgene. Finally, the aqueous layer was lyophilized toobtain a white solid of compound (14) (1.09 g, 98% yield). ¹H NMR (500MHz, D₂O): δ 7.67-7.65 (dd, 2H), 7.50-7.48 (dd, 2H), 3.923 (s, 4H),3.48-2.52 (m, 20H), 1.98 (brs, 2H), 1.88 (brs, 2H); ¹³C NMR (125 MHz,D₂O): S 187.9, 174.4, 173.2, 145.7, 137.5, 136.8, 135.1, 129.1, 128.6,125.5, 122.6, 121.6, 60.4, 59.7, 57.8, 57.2, 56.9, 56.1, 54.5, 48.5,35.5, 29.7, 23.3, 20.7; HRMS (FAB) calculated for C₂₃H₃₆N₅O₄S: 478.2488[(M+H)⁺], measured value: 478.2484 [(M+H)⁺].

Example 5

From compound (4) prepared according to Example 1, functionalizedTE2A-NCS compound (13), to which an isothiocyanate group was introduced,was prepared via the route illustrated in Reaction Scheme 4 below.

Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecane(13) using benzyl 2-bromoacetate Preparation of1,8-bis-(benzyloxycarbonylmethyl)-4-(4′-nitrobenzyl)-1,4,8,11-tetraazacyclotetradecane(15)

To a solution of compound (4) (1.17 g, 2.36 mmol) in dry CHCl₃ (50 ml)were added triethylamine (0.99 ml, 0.72 g, 7.08 mmol) and 4-nitrobenzylbromide (0.51 g, 2.36 mmol). After stirring at an ambient temperaturefor 10 hours, the solvent was removed under reduced pressure, and theresidue was purified through column chromatography on alumina (basic).Extraction with a mixture of acetate and methanol (10:2) gave a purifiedclear oil, which was then solidified to obtain compound (15) (1.13 g,76% yield). ¹H NMR (500 MHz, CDCl₃): δ 8.12-8.10 (dd, 2H), 7.51-7.49(dd, 2H), 7.34-7.27 (m, 10H), 5.11 (s, 2H), 5.05 (s, 2H), 3.55 (s, 2H),3.39 (s, 2H), 3.34 (s, 2H), 2.81 (brs, 2H), 2.74-2.72 (m, 4H), 2.67-2.64(m, 6H), 2.59-2.57 (m, 2H), 2.46-2.44 (m, 2H), 1.71 (brs, 2H), 1.61-1.59(m, 2H); ¹³C NMR (125 MHz, CDCl₃): δ 171.3, 170.8, 148.1, 146.9, 135.7,129.3, 128.5, 128.2, 128.1, 123.3, 66.0, 65.8, 58.1, 55.5, 53.6, 53.1,52.5, 52.0, 51.2, 49.6, 47.9, 47.2, 25.7; HRMS (FAB) calculated forC₃₅H₄₆N₅O₆: 632.3448 [(M+H)⁺], measured value: 632.3447 [(M+H)⁺].

Preparation of1,8-bis-(carboxy)-4-(4′-aminobenzyl)-1,4,8,11-tetraazacyclotetradecane(11)

To a solution of compound (15) (1.08 g, 1.71 mmol) in absolute ethanol(100 ml) was added 5% Pd/CaCO₃ (0.31 g) to which lead (Pb) had beenadded as an inhibitor. The resultant mixture was stirred at an ambienttemperature under H₂ atmosphere for 12 hours. The reaction mixture wasfiltered through a celite pad and washed with ethanol (2×20 ml). Thecombined filtrate was evaporated in vacuo to give an oily residue, whichwas then treated with Et₂O to obtain an off-white solid of compound (11)(0.71 g, 98% yield). ¹H NMR (500 MHz, D₂O): δ 7.20-7.10 (dd, 2H),6.83-6.71 (dd, 2H), 4.1 (s, 2H), 3.52-2.42 (m, 20H), 2.1-1.62 (m, 4H);¹³C NMR (125 MHz, D₂O): δ 192.7, 180.5, 180.4, 149.5, 148.2, 145.3,133.1, 129.8, 128.8, 117.76, 116.4, 116.2, 114.0, 58.3, 57.1, 56.5,55.0, 51.1, 48.8, 46.5, 45.8, 45.4, 23.7, 23.3, 22.7; HRMS calculatedfor C₂₁H₃₆N₅O₄: 422.2767 [(M+H)⁺], measured value: 422.2768 [(M+H)⁺].

Preparation of1,8-bis(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecane(13)

To a solution of compound (11) (0.89 g, 2.11 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (4.85 ml, 7.28 g, 63.3 mmol) inCHCl₃ (10 ml). The reaction mixture was stirred at an ambienttemperature for 5 hours to separate the layers. After removing theaqueous layer, the organic CHCl₃ layer was washed with water (2×50 ml).The combined aqueous layer was washed with CHCl₃ (3×50 ml) to remove theunreacted thiophosgene. Finally, the aqueous layer was lyophilized toobtain a white solid of compound (13) (0.96 g, 98% yield). ¹H NMR (500MHz, D₂O): δ 7.66-7.64 (dd, 2H), 7.49-7.47 (dd, 2H), 4.03 (s, 2H),3.50-2.51 (m, 20H), 2.09 (brs, 2H), 1.87 (brs, 2H); ¹³C NMR (125 MHz,D₂O): δ 192.6, 176.2, 175.7, 133.7, 132.8, 132.0, 123.9, 56.4, 56.2,54.5, 54.0, 51.4, 50.6, 50.2, 49.9, 49.28, 47.7, 45.0, 22.6, 21.7, 13.4;HRMS (FAB) calculated for C₂₂H₃₄N₅O₄S: 464.2332 [(M+H)⁺], measuredvalue: 464.2329 [(M+H)⁺].

Example 6

From compound (4) prepared according to Example 1, functionalizedTE2A-NCS compound (14), to which an isothiocyanate group was introduced,was prepared via the route illustrated in Reaction Scheme 5 below.

Preparation of1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecane(14) using benzyl 2-bromoacetate Preparation of1,8-bis-(benzyloxycarbonylmethyl)-4-(4′-nitrophenethyl)-1,4,8,11-tetraazacyclotetradecane(16)

A solution of compound (4) (1.19 g, 2.39 mmol), 4-nitrophenethyl bromide(1.09 g, 4.78 mmol), anhydrous K₂CO₃ (0.99 g, 7.17 mmol) and KI (1.19 g,7.17 mmol) dissolved in dry toluene (150 ml) was stirred under refluxfor 24 hours. The solvent was evaporated from the reaction mixture underreduced pressure, and CH₂Cl₂ (250 ml) was added thereto. The resultantbrown slurry was filtered through a celite pad, and washed with CH₂Cl₂(2×30 ml). The solvent was evaporated from the combined filtrate underreduced pressure. The residue thus obtained was purified via alumina(basic) column chromatography using EtOAc/methanol (10:2) as an eluentto provide compound (16) as a yellow oil (1.08 g, 70% yield). ¹H NMR(500 MHz, CDCl₃): δ 8.14-8.12 (dd, 2H), 7.49-7.47 (dd, 2H), 7.33-7.28(m, 10H), 5.09 (s, 2H), 5.03 (s, 4H), 3.46 (s, 2H), 3.36 (s, 2H), 3.25(s, 2H), 2.87 (brs, 2H), 2.76-2.72 (m, 4H), 2.65-2.63 (m, 6H), 2.59-2.57(m, 2H), 2.44-2.42 (m, 2H), 1.69 (brs, 2H), 1.62-1.59 (m, 2H); ¹³C NMR(125 MHz, CDCl₃): δ 171.3, 170.8, 148.1, 146.9, 135.7, 129.3, 128.5,128.2, 128.1, 123.3, 66.0, 65.8, 58.1, 55.5, 53.6, 53.1, 52.5, 52.0,51.2, 49.6, 47.9, 47.2, 25.7; HRMS (FAB) calculated for C₃₆H₄₈N₅O₆:646.3605 [(M+H)⁺], measured value: 646.3602 [(M+H)⁺].

Preparation of1,8-bis-(carboxymethyl)-4-(4′-aminophenethyl)-1,4,8,11-tetraazacyclotetradecane(12)

To a solution of compound (16) (0.86 g, 1.33 mmol) in absolute ethanol(100 ml) was added 10% Pd/C (0.26 g). The resultant mixture was stirredat an ambient temperature under H₂ atmosphere for 12 hours. The reactionmixture was filtered through a celite pad and washed with ethanol (2×20ml). The solvent was evaporated from the combined filtrate in vacuo togive an oily residue, which was then treated with Et₂O to obtain a whitesolid of compound (12) (0.57 g, 98% yield). ¹H NMR (500 MHz, D₂O): δ7.45-7.43 (dd, 2H), 7.38-7.36 (dd, 2H), 3.49-3.12 (m, 18H), 2.82-2.62(m, 6H), 1.91 (brs, 4H); ¹³C NMR (125 MHz, D₂O): δ 177.0, 176.6, 163.4,163.1, 162.8, 162.5, 137.4, 130.4, 128.9, 123.4, 119.9, 117.5, 115.2,112.9, 56.0, 54.9, 54.4, 53.3, 51.6, 50.5, 48.8, 47.3, 45.0, 31.5, 27.6,22.8, 21.3; FIRMS (FAB) calculated for C₂₂H₃₈N₅O₄: 436.2924 [(M+H)⁺],measured value: 436.2925 [(M+H)⁺].

Preparation of1,8-bis(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecane(14)

To a solution of compound (12) (0.79 g, 1.81 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (4.17 ml, 6.26 g, 54.4 mmol) inCHCl₃ (10 ml). The reaction mixture was stirred at an ambienttemperature for 5 hours to separate the layers. After removing theaqueous layer, the organic CHCl₃ layer was washed with water (2×50 ml).The combined aqueous layer was washed with CHCl₃ (3×50 ml) to remove theunreacted thiophosgene. Finally, the aqueous layer was lyophilized toobtain a white solid of compound (14) (0.85 g, 98% yield). ¹H NMR (500MHz, D₂O): δ 7.67-7.65 (dd, 2H), 7.50-7.48 (dd, 2H), 3.923 (s, 4H),3.48-2.52 (m, 20H), 1.98 (brs, 2H), 1.88 (brs, 2H); ¹³C NMR (125 MHz,D₂O): δ 187.9, 174.4, 173.2, 145.7, 137.5, 136.8, 135.1, 129.1, 128.6,125.5, 122.6, 121.6, 60.4, 59.7, 57.8, 57.2, 56.9, 56.1, 54.5, 48.5,35.5, 29.7, 23.3, 20.7; HRMS (FAB) calculated for C₂₃H₃₆N₅O₄S: 478.2488[(M+H)⁺], measured value: 478.2484 [(M+H)⁺].

Example 7

Functionalized DO2A(1,7-bis-(carboxymethyl)-1,4,7,10-tetraazacyclododecane)-NCS (compound(24)), to which an isothiocyanate group was introduced, was synthesizedvia the route illustrated in Reaction Scheme 6 below.

Preparation of DO2A-NCS (24) Using tert-butylbromoacetate Preparation of1,7-bis-(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (18)

In a vessel containing ice water, benzyl chloroformate (34.32 ml, 41.59g, 243.8 mmol) was added dropwise to a solution of cyclen compound (17)(20 g, 116.1 mmol) dissolved in CHCl₃ (200 ml). The temperature wasmaintained below 0° C. When the addition was completed, the mixture wasstirred at an ambient temperature for 10 hours so that sufficient solidwas formed. The solvent was then evaporated under reduced pressure toobtain a white solid, to which ether (200 ml) was added. The solid wasfiltered and washed with ether (2×50 ml). Drying in vacuo whilemaintaining the temperature at 45° C. gave dihydrochloride salt (59.01g, 99% yield) as a white solid. To the solid was added 3 M NaOH (250 ml)to obtain the free base. The aqueous layer was extracted with CHCl₃(3×200 ml), and the combined extract was washed with brine and driedover MgSO₄. The solvent was removed by using a rotary evaporator, andthe residue was dried in vacuo for several hours to obtain a solidifiedclear oily compound (18) (50.12 g, 98% yield). ¹H NMR (400 MHz, CDCl₃):δ 7.52-7.32 (m, 10H), 5.18 (s, 4H), 3.83-3.65 (m, 8H), 3.10-2.83 (m,8H); ¹³C NMR (100.6 MHz, CDCl₃): δ 156.5, 136.3, 136.2, 129.0, 128.8,128.7, 128.4, 128.3, 128.2, 68.1, 68.0, 50.9, 50.8, 50.6, 50.5, 50.3,50.0, 49.6, 49.3.

Preparation of1,7-bis-(benzyloxycarbonyl)-4,10-bis(carbo-tert-butoxymethyl)-1,4,7,10-tetraazacyclododecane(19)

To a solution of compound (18) (6.84 g, 15.53 mmol) in dry MeCN (150 ml)were added N,N′-diisopropylethyl amine (13.52 ml, 10.03 g, 77.63 mmol)and tert-butyl bromoacetate (4.82 ml, 6.36 g, 32.61 mmol). The reactionmixture was slowly heated to 60° C., and stirred for 10 hours. Thesolvent was evaporated under reduced pressure, and the residue wasdissolved in a Na₂CO₃ solution (100 ml). The aqueous layer was extractedwith CH₂Cl₂ (3×100 ml), and the combined extract was washed with brineand dried over MgSO₄ to obtain a concentrated white oil. The oil wasrecrystallized from Et₂O to provide compound (19) as a white solid (9.55g, 92% yield). ¹H NMR (500 MHz, CDCl₃): δ 7.26-7.19 (m, 10H), 5.04 (s,4H), 3.34-3.05 (m, 12H), 2.9-2.6 (m, 8H), 1.35 (s, 18H); ¹³C NMR (125MHz, CDCl₃): δ 170.4, 156.3, 136.7, 128.3, 127.8, 127.7, 80.8, 66.8,55.8, 54.2, 46.9, 46.5, 28.1; HRMS (FAB) calculated for C₃₆H₅₃N₄O₈:669.3863 [(M+H)⁺], measured value: 669.3860 [(M+H)⁺].

Preparation of1,7-bis(carbo-tert-butoxymethyl)-1,4,7,10-tetraazacyclododecane (20)

To a solution of compound (19) (8.52 g, 12.74 mmol) in ethanol (130 ml)was added 10% Pd/C (2.6 g). The resultant mixture was stirred at anambient temperature in the presence of H₂ (g) for 12 hours. The reactionmixture was filtered through a celite pad and washed with ethanol (2×20ml). The filtrate was evaporated in vacuo to give an oily residue, whichwas then treated with Et₂O to obtain a white solid of compound (20)(4.85 g, 97% yield). ¹H NMR (500 MHz, CD₃OD): δ 3.44 (s, 4H), 2.91 (s,16H), 1.47 (s, 18H); ¹³C NMR (125 MHz, CD₃OD): δ 173.0, 82.8, 57.4,52.2, 46.5, 28.5; HRMS (FAB) calculated for C₂₀H₄₁N₄O₄: 401.3128[(M+H)⁺], measured value: 401.3132 [(M+¹-1)⁺].

Preparation of1,7-bis-(carbo-tert-butoxymethyl)-4-(4′-nitrobenzyl)-1,4,7,10-tetraazacyclododecane(21)

To a solution of compound (20) (1.85 g, 4.62 mmol) in dry CHCl₃ (50 ml)were added triethylamine (1.93 ml, 1.40 g, 13.86 mmol) and4-nitrobenzylbromide (0.99 g, 4.62 mmol). After stirring the mixture atan ambient temperature for 10 hours, the solvent was removed underreduced pressure, and the residue was purified via alumina (basic)column chromatography. Extraction with ethyl acetate/methanol (10:2)gave a solidified clear oily compound (21) (1.98 g, 80% yield).

Preparation of1,7-bis-(carbo-tert-butoxymethyl)-4-(4′-aminobenzyl)-1,4,7,10-tetraazacyclododecane(22)

To a solution of compound (21) (1.72 g, 3.21 mmol) in absolute ethanol(100 ml) was added 5% Pd/CaCO₃ (0.31 g) to which lead (Pb) had beenadded as an inhibitor. The resultant mixture was stirred at an ambienttemperature in the presence of H₂ (g) for 12 hours. The reaction mixturewas filtered through a celite pad and washed with ethanol (2×20 ml). Thefiltrate was evaporated in vacuo to give an oily residue, which was thentreated with Et₂O to obtain a white solid of compound (22) (1.59 g, 98%yield).

Preparation of1,7-bis-(carboxymethyl)-4-(4′-aminobenzyl)-1,4,7,10-tetraazacyclododecane.2TFA(23.2TFA)

Compound (22) (1.48 g, 2.93 mmol) was dissolved in a mixture of TFA andCH₂Cl₂ (1:1 (v/v), 48 ml). The mixture was stirred at an ambienttemperature for 24 hours. The solvent was removed under reduced pressureto give an oily residue, which was then treated with Et₂O to obtain awhite solid of compound (23) (1.80 g, 99% yield; calculated as 2equivalents of TFA for the basic weight).

Preparation of1,7-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane.2TFA(24.2TFA)

To a solution of compound (23) (1.56 g, 2.51 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (5.77 ml, 8.66 g, 75.30 mmol)dissolved in CHCl₃ (10 ml). The reaction mixture was stirred at anambient temperature for 5 hours, and the resultant layers wereseparated. The aqueous layer was extracted, and the organic CHCl₃ layerwas washed with water (2×50 ml). The combined aqueous layer was washedwith CHCl₃ (3×50 ml) to remove the unreacted thiophosgene. Finally, theaqueous layer was lyophilized to obtain a white solid of compound (24)(1.63 g, 98% yield).

Example 8

Functionalized DO2A-NCS compound (28), to which an isothiocyanate groupwas introduced, was prepared by using tert-butylbromoacetate as anα-halocarboxylic ester (X—CUW—CO₂R⁹) via the route illustrated inReaction Scheme 7 below.

Preparation of DO2A-NCS (28) using tert-butylbromoacetate Preparation of1,7-bis-(carbo-tert-butoxymethyl)-4-(4′-nitrophenethyl)-1,4,7,10-tetraazacyclododecane(25)

A solution of compound (20) (1.56 g, 3.89 mmol), 4-nitrophenethylbromide (1.79 g, 7.78 mmol), anhydrous K₂CO₃ (1.61 g, 11.67 mmol) and KI(1.94 g, 11.67 mmol) dissolved in dry toluene (150 ml) was stirred for24 hours. The solvent was evaporated from the reaction mixture underreduced pressure, and CH₂Cl₂ (250 ml) was added thereto. The resultantbrown slurry was filtered through a celite pad, and washed with CH₂Cl₂(2×30 ml). The solvent was evaporated from the combined filtrate underreduced pressure. The residue thus obtained was purified via alumina(basic) column chromatography using EtOAc/methanol (10:2) as an eluentto provide compound (25) as a yellow oil (1.46 g, 68% yield).

Preparation of1,7-bis-(carbo-tert-butoxymethyl)-4-(4′-aminophenethyl)-1,4,7,10-tetraazacyclododecane(26)

To a solution of compound (25) (1.35 g, 2.46 mmol) in absolute ethanol(100 ml) was added 10% Pd/C (0.41 g). The resultant mixture was stirredat an ambient temperature in the presence of H₂ (g) for 12 hours. Thereaction mixture was filtered through a celite pad and washed withethanol (2×20 ml). The filtrate was evaporated in vacuo to give an oilyresidue, which was then treated with Et₂O to obtain a white solid ofcompound (26) (1.25 g, 98% yield).

Preparation of1,7-bis-(carboxymethyl)-4-(4′-aminophenethyl)-1,4,7,10-tetraazacyclododecane.2TFA(27.2TFA)

Compound (26) (1.12 g, 2.16 mmol) was dissolved in a mixture of TFA andCH₂Cl₂ (1:1 (v/v), 35 ml). The mixture was stirred at an ambienttemperature for 24 hours. The solvent was removed under reduced pressureto give an oily residue, which was then treated with Et₂O to obtain awhite solid of compound (27) (1.37 g, 99% yield; calculated as 2equivalents of TFA for the basic weight).

Preparation of1,7-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,7,10-tetraazacyclododecane.2TFA(28.2TFA)

To a solution of compound (27) (1.25 g, 1.97 mmol) in 0.5 M HCl (10 ml)was carefully added thiophosgene (CSCl₂) (4.53 ml, 6.79 g, 59.10 mmol)dissolved in CHCl₃ (10 ml). The reaction mixture was stirred at anambient temperature for 5 hours, and the resultant layers wereseparated. The aqueous layer was extracted, and the organic CHCl₃ layerwas washed with water (2×50 ml). The combined aqueous layer was washedwith CHCl₃ (3×50 ml) to remove the unreacted thiophosgene. Finally, theaqueous layer was lyophilized to obtain a white solid of compound (28)(1.31 g, 98% yield).

Example 9

A Cu (metal)-coordinated chelate, Cu-TE2A-NO₂ (compound 30), wasprepared from compound (8) obtained according to Example 4 through theroute shown in Reaction Scheme 8 below.

Preparation of Cu-TE2A-NO₂ Chelate Compound (30) Preparation of1,8-bis-(carboxymethyl)-4-(4′-nitrophenethyl)-1,4,8,11-tetraazacyclotetradecane.2TFA(29.2TFA)

Compound (8) (0.95 g, 1.64 mmol) was dissolved in a mixture of CF₃CO₂H(TFA) and CH₂Cl₂ (1:1 (v/v), 35 ml). The resultant mixture was stirredat an ambient temperature for 24 hours. The solvent was evaporated underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain a white solid of compound (29) (1.14 g, 97% yield;calculated as 2 equivalents of TFA for the basic weight). ¹H NMR (500MHz, D₂O): δ 8.14-8.12 (dd, 2H), 7.49-7.48 (dd, 2H), 3.49 (br s, 4H),3.45-2.92 (m, 14H), 2.90-2.61 (m, 6H), 1.97-1.91 (m, 4H); ¹³C NMR (125MHz, D₂O): δ 176.9, 176.6, 146.7, 144.4, 129.9, 124.0, 56.0, 54.9, 54.4,52.9, 51.7, 50.5, 47.4, 45.0, 28.0, 22.9, 21.0; HRMS (FAB) calculatedfor C₂₂H₃₆N₅O₆: 466.2666 [(M+H)⁺], measured value: 466.2661 [(M+H)⁺].

The mass spectrum of compound (29) is shown in FIG. 1.

Preparation of Cu-TE2A-NO₂ Chelate Compound (30)

To a solution of compound (29) (247 mg, 0.36 mmol) and Cu(ClO₄)₂.6H₂O(132 mg, 0.36 mmol) was added an aqueous 1M NaOH solution (2.16 ml). Aclear blue solution thus obtained was heated under reflux for 2 hours.After cooling, the reaction mixture was filtered through a celite pad.The filtered substance was subjected to Et₂O diffusion. The depositedblue crystals were collected and dried to obtain compound (30) (163 mg,87% yield). HRMS (FAB) calculated for C₂₂H₃₃CuNaN₅O₆: 549.1625[(M+Na)⁺], measured value: 549.1629 [(M+Na)⁺]; Visible electronspectrum: λmax (5 M HCl)/561 nm (ε=171 M−1 cm-1)

The mass spectrum of compound (30) is shown in FIG. 2.

Example 10 Acid Decomplexing Experiment of Cu-TE2A-NO₂ Chelate Compound(30)

By using a sample of compound (30) in a concentration of 2.2 mmol in 5 MHCl (2 ml), an acid decomplexing experiment was carried out at 90° C.,under similar initial conditions. Changes in maximum absorption overtime were monitored in a thermostated cell by using a Shimadzu UV-Visspectrophotometer (UV-1650PC). By utilizing the decreasing absorptivityat the λ_(max) of each spectrum (Cu-TE2A 549 nm, Cu-TE2A-NO₂ 561 nm),the progress of the decomplexing reaction was monitored. From the slopeof the linear ln (absorbance) vs. time plots, the half-life wascalculated. Each experiment was repeated 2-3 times, and the averagehalf-life was obtained. The measured results are shown in Table 2 andFIGS. 3A and 3B.

Example 11 Electrochemical Experiment for Cu-TE2A-NO₂ Chelate Compound(30)

Cyclic voltammetry was carried out by using biological model SP-150having a 3-electrode structure. The working electrode was made of glassycarbon (diameter=3 mm), the reference electrode Ag/AgCl (saturated KCl),and the counter electrode Pt wire. A sample (2 mM) of compound (30) wasoperated at a scanning speed of 100 mV/s in a 0.1 M aqueous sodiumacetate solution adjusted to pH 7.0 with glacial acetic acid. From thesolution, oxygen was removed by bubbling Ar for 30 minutes. During themeasurement, the system was maintained under Ar atmosphere. The measuredresults are shown in Table 2 and FIGS. 4A and 4B.

TABLE 2 Sample Half-life (5M HCl, 90° C.) E_(red) (V) for Ag/AgClCu-TE2A 46.2 min −1.05 (irrev) Compound (30) 47.7 min −0.98 (irrev)

The kinetic inertness and reduction potential of compound (30) werenearly identical to those of Cu-TE2A. Based on those results, it can berecognized that the introduction of a third orthogonal pendant armhaving a NCS functional group for conjugation with a peptide or anantibody to the TE2A backbone does not inhibit higher kinetic inertnessthan a TETA (half-life: 4.7 min) analogue that has been mostconventionally used.

Example 12

From compound (5) according to Example 2, TE2A-mono-Me (compound 32), towhich a methyl group was introduced, was prepared via the route shown inReaction Scheme 9 below.

Preparation of1,8-bis-(carboxymethyl)-4-(methyl)-1,4,8,11-tetraazacyclotetradecane.2TFA(32.2TFA) Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4-methyl-1,4,8,11-tetraazacyclotetradecane(31)

To a solution of compound (5) (2.33 g, 5.43 mmol) in dry chloroform (50ml) was added methyl iodide (6.78 ml, 15.43 g, 108.72 mmol). Afterstirring at an ambient temperature for 24 hours, the solvent was removedfrom the reaction mixture under reduced pressure. The residue waspurified via column chromatography on silica using chloroform/isopropylamine (20:2) as an eluent, to obtain compound (31) as a clear oil (2.41g, 84% yield). ¹H NMR (500 MHz, CDCl₃): δ 3.27-3.25 (dd, 4H), 2.84-2.43(m, 16H), 2.16 (s, 3H), 1.73-1.59 (m, 4H), 1.45 (s, 18H); ¹³C NMR (125MHz, CDCl₃): δ 170.94, 170.68, 80.57, 55.99, 55.93, 54.80, 53.77, 53.40,52.26, 50.11, 48.34, 47.41, 47.17, 41.88, 28.18, 25.59, 25.00; HRMS(FAB) calculated for C₂₃H₄₇N₄O₄: 443.3597 [(M+H)⁺], measured value:443.3600 [(M+H)⁺].

The mass spectrum of compound (31) is shown in FIG. 5.

Preparation of1,8-bis-(carboxymethyl)-4-(methyl)-1,4,8,11-tetraazacyclotetradecane.2TFA(32.2TFA)

Compound (31) (1.56 g, 3.52 mmol) was dissolved in a mixture of CF₃CO₂H(TFA) and CH₂Cl₂ (1:1 (v/v), 60 ml). The resultant mixture was stirredat an ambient temperature for 24 hours. The solvent was removed underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain a white solid of compound (32) (1.95 g, 99% yield;calculated as 2 equivalents of TFA for the basic weight). ¹H NMR (500MHz, D₂O): δ 3.60-3.05 (m, 13H), 2.98-2.641 (m, 10H), 2.12-1.82 (m, 4H);¹³C NMR (125 MHz, D₂O): δ 177.20, 175.83, 56.71, 55.87, 54.50, 54.22,52.92, 48.268, 44.94, 41.15, 22.61, 20.64; HRMS (FAB) calculated forC₁₅H₃₁N₄O₄: 331.2345 [(M+H)⁺], measured value: 331.2347 [(M+H)⁺].

The mass spectrum of compound (32) is shown in FIG. 6.

Example 13

From compound (3) according to Example 1, TE2A-di-Me (compound 34), towhich two methyl groups were introduced, was prepared via the routeshown in Reaction Scheme 10 below.

Preparation of1,8-bis-(carboxymethyl)-4,11-bis-(methyl)-1,4,8,11-tetraazacyclotetradecane(34) Preparation of1,8-bis-(carbo-tert-butoxymethyl)-4,11-bis-(methyl)-1,4,8,11-tetraazacyclotetradecane(33)

To a solution of compound (3) (3.06 g, 7.14 mmol) in absolute ethanol(80 ml) was added NaBH₄ (8.10 g, 214.2 mmol). After stirring at anambient temperature for 24 hours, the solvent was removed under reducedpressure. The residue was dissolved in CH₂Cl₂ (150 ml), and filtered.The filtered substance was dried, and the residue was purified viacolumn chromatography on silica using chloroform/isopropyl amine (20:2)as an eluent, to obtain compound (33) as a clear oil (3.05 g, 94%yield). ¹H NMR (500 MHz, CDCl₃): δ 3.23 (s, 4H), 2.80-2.62 (m, 8H), 2.43(br s, 8H), 2.19 (s, 6H), 1.68-1.58 (m, 4H), 1.42 (s, 18H); ¹³C NMR (125MHz, CDCl₃): δ 170.89, 80.61, 56.50, 54.51, 53.91, 51.00, 50.55, 43.29,28.15, 24.66; HRMS (FAB) calculated for C₂₄H₄₉N₄O₄: 457.3754 [(M+H)⁺],measured value: 457.3756 [(M+H)⁺].

The mass spectrum of compound (33) is shown in FIG. 7.

Preparation of1,8-bis-(carboxymethyl)-4,11-bis-(methyl)-1,4,8,11-tetraazacyclotetradecane(34)

Compound (33) (1.46 g, 3.19 mmol) was dissolved in a mixture of CF₃CO₂H(TFA) and CH₂Cl₂ (1:1 (v/v), 50 ml). The resultant mixture was stirredat an ambient temperature for 24 hours. The solvent was removed underreduced pressure to give an oily residue, which was then treated withEt₂O to obtain a white solid of compound (34) (1.09 g, 99% yield). HRMS(FAB) calculated for C₁₆H₃₃N₄O₄: 345.2502 [(M+H)⁺], measured value:345.2506 [(M+H)⁺].

The mass spectrum of compound (34) is shown in FIG. 8.

As shown in Example 13, according to the present invention, compounds inwhich substituents have been symmetrically introduced to amine, such ascompound (34), as well as compounds in which substituents have beenasymmetrically introduced to amine, such as compound (32), can besynthesized.

Example 14

A Cu-chelated compound, Cu-TE2A-mono-Me (compound 35), was prepared fromcompound (32) obtained according to Example 12 via the route shown inReaction Scheme 11 below.

Preparation of Cu-TE2A-mono-Me Chelate Compound (35)

To a solution of compound (32) (265 mg, 0.47 mmol) and Cu(ClO₄)₂.6H₂O(176 mg, 0.47 mmol) in 22 ml of methanol was added an aqueous 1M NaOHsolution (2.82 ml). A blue solution thus obtained was heated underreflux for 2 hours. After cooling, the reaction mixture was filteredthrough a celite pad. The filtered substance was subjected to Et₂Odiffusion. The deposited blue crystals were collected and dried toobtain compound (35) (166 mg, 89% yield). HRMS (FAB) calculated forC₁₅H₂₈CuNaN₄O₄: 414.1304 [(M+Na)⁺], measured value: 414.1302 [(M+Na)⁺].

The mass spectrum of compound (35) is shown in FIG. 9.

Example 15

A Cu-chelated compound, Cu-TE2A-di-Me (compound 36), was prepared fromcompound (34) obtained according to Example 13 via the route shown inReaction Scheme 12 below.

Preparation of Cu-TE2A-di-Me Chelate Compound (36)

To a solution of compound (34) (253 mg, 0.73 mmol) and Cu(ClO₄)₂.6H₂O(272 mg, 0.73 mmol) in 25 ml of methanol was added an aqueous 1M NaOHsolution (4.38 ml). A blue solution thus obtained was heated underreflux for 2 hours. After cooling, the reaction mixture was filteredthrough a celite pad. The filtered substance was subjected to Et₂Odiffusion. The deposited blue crystals were collected and dried toobtain compound (36) (253 mg, 85% yield). HRMS (FAB) calculated forC₁₆H₃₀CuNaN₄O₄: 428.1461 [(M+Na)⁺], measured value: 428.1462 [(M+Na)⁺].

The mass spectrum of compound (36) is shown in FIG. 10.

Example 16

A metal chelating conjugate compound can be prepared by binding BFC to abioactive molecule such as a peptide, and chelating the metal, or bybinding a bioactive molecule such as a peptide to a metal-BFC chelate(which was prepared in advance). In the present Example,[⁶⁴Cu-TE2A-c(RGDyK)] metal chelating conjugate compound (38) wasprepared by binding TE2A-NCS compound (14) (prepared according to theExample described above) via the route shown in Reaction Scheme 13 to apeptide c(RGDyK) to provide a conjugate compound, and chelating themetal ⁶⁴Cu.

Preparation of [⁶⁴Cu-TE2A-c(RGDyK)] metal chelating conjugate compound(38) Preparation of a conjugate compound of TE2A-NCS and peptidec(RGDyK) (37)

A solution of TE2A-NCS compound (14) (495 nmol, 2.36 mg) was combinedwith peptide c(RGDyK) (165 nmol, 1.02 mg) in 0.1 M Na₂CO₃ buffer (pH9.5). Under a light-shielded environment, the mixture was stirred at anambient temperature for 22 hours, and subjected to semi-preparative highperformance liquid chromatography (HPLC) (Agilent preparative columnC18; 5 μm, 21.2×100 mm; flow rate 3 ml/min, mobile phase: starting with95% solvent A [aqueous 0.1% TFA solution] and 5% solvent B [0.1% TFA inMeCN] [0-2 min] to 35% solvent A and 65% solvent B at 32 min), toisolate the c(RGDyK) peptide conjugated to TE2A. At the 21.7 minretention time on the HPLC, visible TE2A-c(RGDyK) was collected andlyophilized to provide TE2A-c(RGDyK) compound (37) as a white powder(82% yield). On an analytical HPLC column (Vydac TP C18; 3 μm, 4.6×100mm; flow rate 1 ml/min, mobile phase: 0.1% TFA/H₂O (solvent A) and 0.1%TFA/MeCN (solvent B), and a gradient elution of 1% B to 70% B in 20minutes), the retention time of TE2A-c(RGDyK) compound (37) was 12.8min. The purified TE2A-c(RGDyK) compound (37) was identified by using ajet-type mass analyzer (m/z calculated for C₅₀H₇₇N₁₄O₁₂S was 1097.55,m/z affirmed for [MH]⁺ and [MH₂]⁺²: 1097.58 and 549.62, respectively).

FIG. 11 shows a chromatogram of TE2A-c(RGDyK) compound (37) onsemi-preparative HPLC, while FIG. 12 shows a chromatogram ofTE2A-c(RGDyK) compound (37) on analytical HPLC. FIG. 13 shows a massspectrum of TE2A-c(RGDyK) compound (37).

Preparation of [⁶⁴Cu-TE2A-c(RGDyK)] metal chelating conjugate compound(38)

To TE2A-c(RGDyK) compound (37) (2 μg) in 100 μl of 0.1 M NH₄OAc buffer(pH 8.0) was added ⁶⁴Cu (0.52 mCi) in 100 μl of 0.1 M NH₄OAc buffer (pH8.0). The mixture was reacted at 30° C. for 5 minutes. The reaction wasmonitored through radio-TLC using Whatman MKC18F thin layerchromatography (TLC) plate developed by 10% NH₄OAc/methanol (30:70)[R_(f) of ⁶⁴Cu-TE2A-c(RGDyK)=0.9]. The ⁶⁴Cu-labeled peptide was furtherpurified via reverse-phase (RP) HPLC [Vydac TP C18; 3 μm, 4.6×100 mm;flow rate 1 ml/min, mobile phase: 0.1% TFA/H₂O (solvent A) and 0.1%TFA/MeCN (solvent B), and a gradient elution of 1% B to 70% B in 20minutes]. After collecting ⁶⁴Cu-TE2A-c(RGDyK) compound (38) (retentiontime [t_(R)]: 13.8 min) by using 12 ml of the HPLC solvent, the solventwas evaporated and the residue was recovered with PBS(phosphate-buffered saline). Then the recovered ⁶⁴Cu-TE2A-c(RGDyK)compound (38) was filtered through a 0.22 μm Millipore filter, andtransferred to a sterile bottle for animal tests.

FIG. 14 shows a radio-TLC chromatogram of ⁶⁴Cu-TE2A-c(RGDyK) compound(38), while FIG. 15 shows a radio chromatogram of ⁶⁴Cu-TE2A-c(RGDyK)compound (38) on analytical HPLC. FIG. 16 simultaneously showsTE2A-c(RGDyK) compound (37) and ⁶⁴Cu-TE2A-c(RGDyK) compound (38) onanalytical HPLC, in order to confirm the preparation of the metalchelating conjugate.

Example 17 Experiment for In Vivo Distribution of ⁶⁴Cu-TE2A-c(RGDyK)

Compound ⁶⁴Cu-TE2A-c(RGDyK) (38) (10 μCi) in PBS (120 μl) was injectedto the tails of female nude mice to which U87MG tumor had beentransplanted. Two groups were examined at two time points [n=4 per groupat 1 hr and 4 hr post injection (pi)]. The subject animals weresacrificed, and the relevant tissues and organs were removed andweighed. A dosimetric procedure was carried out by using agamma-counter. The calculations were performed by comparing with areference value of which the percentage of injected amount per gram wasknown. The test results (% ID/g±SD, n=4) are shown in Table 3 and FIG.17.

TABLE 3 Tissue 1 hr 4 hr Blood 0.55 ± 0.18 0.43 ± 0.09 Lung 1.46 ± 0.341.14 ± 0.17 Muscle 0.49 ± 0.30 0.24 ± 0.11 Fat 1.28 ± 0.55 1.15 ± 1.08Bone 0.66 ± 0.23 0.41 ± 0.06 Spleen 1.17 ± 0.46 1.04 ± 0.46 Kidney 3.47± 0.61 2.71 ± 0.45 Liver 5.45 ± 1.14 4.45 ± 0.65 Stomach 1.78 ± 0.551.03 ± 0.39 Intestine 2.14 ± 0.53 2.17 ± 0.62 Tumor 1 2.98 ± 0.39 3.01 ±1.00 Tumor 2 3.49 ± 1.67 3.32 ± 0.49

Example 18 Micro PET Image Analysis of ⁶⁴Cu-TE2A-c(RGDyK)

PET scans and image analyses of the present Example were carried out byusing a Micro PET R4 rodent model scanner. The imaging study was carriedout with a female nude mouse bearing 41-days U87MG tumors. Compound⁶⁴Cu-TE2A-c(RGDyK) (38) (205 μCi) was injected to the tail of the mouse.After 1 hour, 4 hours, 1 day, 2 days and 3 days after injection, themouse was anesthetized with 1-2% isoflurane. The mouse was fixed lyingits face down, and an image was obtained. The images were reconstitutedby an algorithm of 2-dimensional ordered subsets expectationmaximization (OSEM), without any correction of attenuation orscattering.

FIG. 18 shows the administration of ⁶⁴Cu-TE2A-c(RGDyK) compound (38) tothe subject animal, a female nude mouse having U87MG tumor cells. FIG.19 shows Micro PET images over time (at 1 hour, 4 hours, 1 day, 2 daysand 3 days) after administration of ⁶⁴Cu-TE2A-c(RGDyK) compound (38).

Example 19

In the present Example, [⁶⁴Cu-TE2A-trastuzumab] metal chelatingconjugate compound (40) was prepared via the route shown in ReactionScheme 14 below, by binding TE2A-NCS compound (14) obtained according tothe Example above to an antibody trastuzumab (Herceptin), and chelating⁶⁴Cu metal thereto.

Preparation of [⁶⁴Cu-TE2A-trastuzumab] Metal Chelating ConjugateCompound (40) Preparation of TE2A-NCS and Antibody Trastuzumab ConjugateCompound (39)

To trastuzumab (2 mg) was added a 50-fold excessive amount of TE2A-NCScompound (14) (0.33 mg) in 0.1 M Na₂CO₃ (pH 9.5, 100 μl). The solutionwas gently stirred at an ambient temperature for 24 hours. One daylater, the content was transferred to Centricon YM-50, which wascentrifuged to decrease the solvent. To the resultant TE2A-trastuzumabwas added PBS (pH 7.2, 3×2 ml), and the content was centrifuged toremove the unreacted ligand. To the purified TE2A-trastuzumab compound(39) was added PBS 2.0 ml, and the mixture was maintained at −20° C.

Preparation of [⁶⁴Cu-TE2A-trastuzumab] Metal Chelating ConjugateCompound (40)

To TE2A-trastuzumab compound (39) (50 μg) in 0.1 M NH₄OAc buffer (pH8.0) (100 μl) was added ⁶⁴Cu (0.52 mCi) in 0.1 M NH4OAc buffer (pH 8.0).The solution was reacted at 30° C. for 5 minutes. The ⁶⁴Cu-labeledTE2A-trastuzumab was purified by centrifugation with Microcon YM-50. Theradiochemical purity was identified by size exclusion chromatography(SEC) HPLC (BioSilect SEC 250-5 300×7.8 mm; flow rate 1 ml/min, with theisocratic mobile phase consisting of PBS, pH=7.4) and instant TLC(ITLC-SG, developed by saline).

FIG. 20 shows a radio-ITLC chromatogram of ⁶⁴Cu-TE2A-trastuzumabcompound (40), while FIG. 21 simultaneously shows chromatograms ofTE2A-trastuzumab compound (39) and ⁶⁴Cu-TE2A-trastuzumab compound (40)on SEC HPLC, in order to confirm the preparation of the metal chelatingconjugate compound.

Example 20 Experiment of Distribution In Vivo of ⁶⁴Cu-TE2A-Trastuzumab

Compound ⁶⁴Cu-TE2A-trastuzumab (40) (20 μCi) in PBS 120 μl was injectedto the tails of female nude mice to which NIH3T6.7 tumor had beentransplanted. Two groups were examined at two time points [n=4 per groupat 1 day and 2 days post injection (pi)]. The subject animals weresacrificed, and the relevant tissues and organs were collected andweighed. A dosimetric procedure was carried out by using agamma-counter. The calculations were performed by comparing with areference value of which the percentage of injected amount per gram wasknown. The test results (% ID/g±SD, n=4) are shown in Table 4 and FIG.22.

TABLE 4 Tissue/organ Day 1 Day 2 Blood 21.91 ± 3.74  22.56 ± 9.60  Heart4.67 ± 2.11 4.64 ± 1.47 Lung 8.28 ± 1.73 9.73 ± 3.06 Muscle 3.81 ± 0.374.94 ± 1.84 Bone 4.53 ± 1.11 4.60 ± 1.46 Spleen 6.51 ± 2.00 8.43 ± 1.77Kidney 8.15 ± 1.81 9.56 ± 1.80 Stomach 2.18 ± 0.97 2.70 ± 0.64 Intestine3.16 ± 0.65 3.56 ± 0.74 Liver 10.67 ± 2.54  11.50 ± 2.25  Tumor (R)20.85 ± 9.57  26.34 ± 9.05  Tumor (L) 25.65 ± 6.54  25.86 ± 10.23

Example 21 Micro PET Image Analysis of ⁶⁴Cu-TE2A-trastuzumab

PET scans and image analyses of the present Example were carried out byusing a Micro PET R4 rodent model scanner. The imaging test was carriedout with a female nude mouse bearing 31-days NIH3T6.7 tumors. Compound⁶⁴Cu-TE2A-trastuzumab (40) (145 μi) was injected to the tail of themouse. After 1 hour, 4 hours, 1 day, 2 days, 3 days and 5 days afterinjection, the mouse was anesthetized with 1-2% isoflurane. The mousewas fixed with its face down, and an image was obtained. The images werereconstituted by an algorithm of 2-dimensional OSEM, without anycorrection of attenuation or scattering.

FIG. 23 shows the administration of ⁶⁴Cu-TE2A-trastuzumab compound (40)to the subject animal, a female nude mouse having NIH3T6.7 tumor cells.FIG. 24 shows Micro PET images at 1 hour, 4 hours, 1 day, 2 days, 3 daysand 5 days after administration of ⁶⁴Cu-TE2A-trastuzumab compound (40).

The various examples described above are not intended to restrict thesubject of the present invention, of which authentic scope and purposeare indicated by the claims attached.

What is claimed is:
 1. A polyazamacrocyclic compound represented byChemical Formula 1 or a pharmaceutically acceptable salt thereof:

wherein m, n, p and q are identical to or different from one another,and individually represent an integer of 2 or 3, with the proviso thatm, n, p and q are not all two, r is an integer from 0 to 5, t is aninteger of 0 or 1, r+t>0, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areidentical to or different from one another, and individually representH, C₁₋₅ alkyl or C₃₋₆ cycloalkyl, R¹⁰ represents H, C₁₋₅ alkyl, C₃₋₆cycloalkyl, or C₇₋₁₄ aralkyl, U and W are identical to or different fromone another, and individually represent H, C₁₋₅ alkyl or C₃₋₆cycloalkyl, Y and Z are identical to or different from one another, andindividually represent H, C₁₋₅ alkyl or C₃₋₆ cycloalkyl, A representsC₆₋₁₀ aryl, Q represents H, nitro, amino, isothiocyanato, maleimido,alkyne, aminoxy, thiol, or azide.
 2. The polyazamacrocyclic compound orpharmaceutically acceptable salt thereof according to claim 1, whereinthe pharmaceutically acceptable salt, when the compound represented byChemical Formula 1 contains a negatively charged component, comprises acation or a cationic group selected from the group consisting ofpotassium, sodium, lithium, ammonium, silver, calcium and magnesium, orwhen the compound represented by Chemical Formula 1 contains apositively charged component, comprises an anion or an anionic groupselected from the group consisting of F⁻, Cl⁻, Br⁻, I⁻, ClO₄ ⁻, BF₄ ⁻,HCO₃ ⁻, CH₃CO₂ ⁻, CH₃SO₃ ⁻, CH₃C₆H₄SO₃ ⁻, CF₃SO₃ ⁻, H₂PO₄ ⁻ and B(C₆H₅)₄⁻.
 3. The polyazamacrocyclic compound or pharmaceutically acceptablesalt thereof according to claim 1, wherein Q is isothiocyanato.
 4. Thepolyazamacrocyclic compound or pharmaceutically acceptable salt thereofaccording to claim 1, wherein Q is amino.
 5. The polyazamacrocycliccompound or pharmaceutically acceptable salt thereof according to anyone of claims 1 to 3, which is1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatobenzyl)-1,4,8,11-tetraazacyclotetradecane,1,8-bis-(carboxymethyl)-4-(4′-isothiocyanatophenethyl)-1,4,8,11-tetraazacyclotetradecane,1,8-bis-(carboxymethyl)-4-(4′-nitrophenethyl)-1,4,8,11-tetraazacyclotetradecane,or 1,8-bis-(carboxymethyl)-4-(methyl)-1,4,8,11-tetraazacyclotetradecane.6. A method for preparing a polyazamacrocyclic compound represented byChemical Formula 1, which comprises the steps of (i) reacting a compoundrepresented by Chemical Formula 9 with α-halocarboxylic ester(X—CUW—CO₂R⁹) to obtain a trans-N,N′-disubstituted compound representedby Chemical Formula 10, (ii) reacting a compound represented by ChemicalFormula 10 with a base to obtain a compound represented by ChemicalFormula 11, (iii) introducing a functional group —(CYZ)_(r)-A_(t)-Q to asecondary amine group in the cycle of compound represented by ChemicalFormula 11 to form a compound represented by Chemical Formula 1:

wherein m, n, p and q are identical to or different from one another,and independently represent an integer of 2 or 3, with the proviso thatm, n, p and q are not all two, r represents an integer from 0 to 5, trepresents an integer of 0 or 1, r+t>0, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸are identical to or different from one another, and independentlyrepresent H, C₁₋₅ alkyl or C₃₋₆ cycloalkyl, R⁹ represents C₁₋₅ alkyl,C₃₋₆ cycloalkyl, or C₇₋₁₄ aralkyl, R¹⁰ represents H, C₁₋₅ alkyl, C₃₋₆cycloalkyl, or C₇₋₁₄ aralkyl, U and W are identical to or different fromone another, and individually represent H, C₁₋₅ alkyl or C₃₋₆cycloalkyl, X represents F, Cl, Br or I, Y and Z are identical to ordifferent from one another, and individually represent H, C₁₋₅ alkyl orC₃₋₆ cycloalkyl, A represents C₆₋₁₀ aryl, and Q represents H, nitro,amino, isothiocyanato, maleimido, alkyne, aminoxy, thiol, or azide. 7.The method according to claim 6 for preparing a polyazamacrocycliccompound of Chemical Formula 1 wherein the α-halocarboxylic ester istert-butylbromoacetate or benzyl bromoacetate.